DNA Extraction Methods PDF

Dna eXTRACTION  INTRODUCTION DNA extraction is one of the basic techniques employed in the molecular laboratory, done for the first time by Friedrich Miescher in 1869. Although Friedrich Miescher isolated the cell material “nuclei” or “nucleic aci...

Dna eXTRACTION  INTRODUCTION DNA extraction is one of the basic techniques employed in the molecular laboratory, done for the first time by Friedrich Miescher in 1869. Although Friedrich Miescher isolated the cell material “nuclei” or “nucleic acid” and developed a method for nucleic acid isolation, he was not sure whether what he isolated was DNA or not. In 1958 Stahl and Meselson developed a full- function protocol for DNA extraction (The density gradient centrifugation protocol) that was the first protocol described for isolating DNA from E.coli bacteria.  what is DNA Extraction ? DNA extraction is a process that used to purify DNA by using chemical or physical methods from a biological sample separating DNA from protein, cell membranes, and other cellular components. DNA extraction is used to isolate… Mitochondrial DNA Genomic DNA DNA can be extracted from almost any intact cellular tissue Skin, blood, saliva, semen, mucus, muscle tissue, bone marrow, etc.  What is DNA Extraction Principle? DNA extraction involves lysis of cellular and nuclear membranes in order to extract DNA from within. This is followed by DNA separation from impurities, proteins, and other substances.  General Stages of DNA Extraction are: 1 2 2 Cell dissolution Precipitation Purification  Basic step of DNA extraction 1. Lysis of cell wall or cell membrane. 2. Lysis of nuclear membrane. 3. Extracting the DNA from the other cell debris 4. DNA precipitation 5. Dissolving DNA  Note The main steps remain similar among all DNA extraction methods.  DNA extraction methods are broadly categorized into: Chemical-based (or solution- based) DNA extraction methods. Solid-phase DNA extraction methods (Physical method).  Chemical-based DNA extraction method The Chemical or solution-based method uses many organic and inorganic solutions. Chemicals like phenol, chloroform, CTAB Triton X100, SDS, isoamyl alcohol, Tris and EDTA are used in the chemical -based DNA extraction method.  chemical-based DNA extraction method is subdivided into: Inorganic solvent- Organic solvent-based based DNA extraction DNA extraction This method depends on the It depends on the inorganic use of organic substances solvents. such as phenol and chloroform. Example: Salting out method, SDS DNA Example: Phenol-chloroform and isoamyl alcohol extraction, CTAB DNA extraction, Silica-gel-based techniques Physical or Solid-Based DNA Extraction Methods Paper DNA extraction Magnetic bead DNA extraction  Liquid-Liquid DNA Extraction Liquid-liquid extraction is one of the commonest methods for nucleic acid extraction. In this method the solutions prepared by various chemical compositions are used for extraction and it mainly relies on lysis buffer preparation. These methods commonly require centrifugation for separation. Examples of liquid-liquid DNA extraction methods: Phenol, chloroform and isoamyl alcohol DNA extraction, SDS DNA extraction and CTAB DNA extraction.  Although this method is considered one of the best methods, it is less recommended because of phenol and chloroform harmful. The quantity and quality of DNA obtained by method are very good. This method is known as a phenol-chloroform and isoamyl alcohol or PCI method of DNA extraction. Unfortionatly, this method is it is unsafe because Phenol is volatile and can cause burns. the chloroform can faint us. So, it requires training and to prepare and handle chemicals. ORGANIC EXTRACTION Phenol-Chloroform Method  ORGANIC EXTRACTION PROCEDURE Typical Procedure 1. Cell Lysis 2. Phenol Extraction 3. RNAse followed by proteinase K 4. Repeat Phenol Extraction 5. Ethanol Precipitation  ORGANIC EXTRACTION REAGENTS Proteinase K - it is usual to remove most of the protein by digesting with proteolytic enzymes such proteinase K Phenol and chloroform are used to denature and separate proteins from DNA. Phenol denatures proteins and dissolves denatured proteins. Chloroform is also a protein denaturant DNA released from disrupted cells is precipitated by cold absolute ethanol or isopropanol. Non-Organic DNA Extraction The Salting-out DNA Extraction  Salting-out extraction method is a simple and non-toxic technique that isolates a high-quality DNA from the whole blood. In the standard salting-out method, proteins K and RNase are added to them after the lysis of cells. Saturated NaCl was needed for the proteins to precipitate out of the solution. The cell samples are centrifuged and then, DNA is separated by washing it with detergent like ethanol.  The salting-out DNA extraction method is safer than the phenol-chloroform method. It depends on the usage of salts such as potassium acetate, sodium chloride, and ammonium acetate that help in DNA extraction. This method gives excellent results in combination with proteinase K and enough yield can be obtained but the quality (purity) obtained might not be good.  SDS DNA Extraction SDS or sodium dodecyl sulfate is an anionic detergent that digests nuclear and cell membrane proteins. SDS gives a negative charge to each protein as a function of their size. So, SDS can be used to aid in lysing cell during DNA extraction.  CTAB DNA Extraction CTAB or Cetyl Trimethyl Ammonium Bromide is a chemical often used in DNA extraction. This method is a specially prepared liquid-liquid and solution- based extraction method for plant DNA extraction. The CTAB buffer removes polysaccharides and polyphenols effectively and gives excellent yield for plant DNA. This method required extensive chemical preparation and additional techniques like tissue homogenization and the use of liquid nitrogen. It also takes a long time.  Silica Gel Based Techniques Spin column is the most advanced, effective, rapid and accurate separation technique. It is, however, a chemical method of DNA extraction but works on the principle of solid-phase separation.  The main advantage of this technique is that can isolate DNA from any biological sample and tissue. However, it is difficult to isolate DNA from plants even using the spin-column technique, Different columns are now available depending upon the sample type. But this technique is Cost, lacks optimizations and the yield remains comparatively low but good.  Advantages of DNA Extraction Methods  DNA extraction is vital to biology, especially biotechnology. It is the first step of different applications like fundamental research, disease diagnosis and therapeutic decision. One of the main advantages of DNA extraction methods is that they are very importance to define the unique characteristics of DNA like the shape, the size and function. DNA helped in finding out the molecular basis and cure for various diseases.  In criminal investigations, DNA extraction from samples (e.g., hair – skin – blood) is used to determine if a person is a suspect or not and also it can prove whether a person was in the vicinity of the crime scene. It also used in Paternity Tests. It can be useful in genetic engineering. For animals, DNA extraction is helpful for transforming and cloning animal’s DNA. For plants, DNA can be useful in identifying and extracting a specific gene in order to replicate in generations of plants. DNA study also helped in creating many vaccines (e.g., Hepatitis B vaccine), hormones (e.g., growth hormones and insulin), and enzymes.  How do I determine the concentration, yield and purity of a DNA sample? DNA yield can be assessed using various methods including absorbance (optical density), agarose gel electrophoresis, or use of fluorescent DNA-binding dyes. All three methods are convenient, but have varying requirements in terms of equipment needed, ease of use, and calculations to consider.  Absorbance Methods The most common technique to determine DNA yield and purity is measurement of absorbance. In the case of nucleic acid (DNA and RNA), the maximal absorbance is at 260nm. Protein maximally absorbs at 280nm and the ratio of nucleic acid to protein (260/280) is generally used as an indicator of the purity of DNA samples.  Spectrophotometer Tips ✓ Before measuring any samples, be sure to ‘blank’ the spectrophotometer using the solution the DNA is resuspended in, but with no DNA added. ✓ If using a NanoDrop to measure your samples, place 1-2µL of mini- prepped DNA onto the pedestal. ✓ Close the lid and click measure, be sure to record the concentration and purity. ✓ Note: Purity is measured under the 260/280 column (A good purity ranges from 1.80-2.00). Concentration (µg/ml) = (A260 reading – A320 reading) × dilution factor × 50µg/ml or Concentration (ng/ml) = A260 reading x dilution factor x 50 ng/ml DNA yield (µg) = DNA concentration × total sample volume (ml) Exercise # 1 Calculate the concentration of DNA ([DNA]) in the sample using the data shown below: The DNA sample was diluted 1/5. The Absorbance at 260 nm of the diluted solution was 0.25 Exercise # 2 What will be the Absorbance at 260 nm of a diluted solution (1\10) if the concentration of DNA in the sample is 50 mg/ mL? Exercise # 3 Calculate the total amount of DNA in the sample using the data shown below: Total volume of the DNA sample was 200 mL. The DNA sample was diluted 1/5. Absorbance at 260 nm of the diluted solution was 0.25  Fluorescence Methods The widespread availability of fluorometers and fluorescent DNA- binding dyes makes fluorescence measurement another popular option for determining of DNA yield and concentration. Fluorescence methods are more sensitive than absorbance, particularly for low-concentration samples, and the use of DNA- binding dyes allows more specific measurement of DNA than spectrophotometric methods allows.  Agarose Gel Electrophoresis Concentration and yield can be determined after gel electrophoresis is completed by comparing the sample DNA intensity to that of a DNA quantitation standard. For example, if a 2µl sample of undiluted DNA loaded on the gel has the same approximate intensity as the 100ng standard, then the solution concentration is 50ng/µl (100ng divided by 2µl). Thank You